Peptide thioneamides as selective substrates for cysteine proteases

ABSTRACT

Peptide thioneamides are provided as synthetic substrates for cysteine proteases. The compounds of the invention are peptides consisting of one or more blocked or unblocked amino acids wherein the terminal carboxy of the peptides are thionated and forms a thioneamide linkage with a fluorogenic or chromogenic leaving group. The alpha conbonyls of the remaining amino acids are thionated or unthionated. The invention includes methods and kits for using the peptide thioneamides.

BACKGROUND OF THE INVENTION

This invention relates to synthetic substrates and their use fordetermining the activities of enzymes, and more specifically, to peptidethioneamides and their use for determining the activities of cysteineproteases.

The determination of specific enzymes in biological fluids, such asblood, tissue homogenates, urine, or the like, is very useful for thediagnosis of certain diseases, e.g. Evered et al., eds. ProteinDegradation in Health and Disease (Exerpta Medica, Amsterdam, 1980).Synthetic substrates have been developed and utilized for suchdeterminations, resulting in clinical assay procedures having a highdegree of specificity, reliability, and sensitivity. Syntheticsubstrates have generally been amino acid or peptide derivativesacylated to aromatic amines, the latter becoming fluorimetrically orspectrophotometrically detectable after being cleaved from the aminoacid or peptide, e.g. see Lorand, ed.,"Proteolytic Enzymes," Methods inEnzymology, Vol. 80 (Academic Press, New York, 1981). The number andordering of amino acids in the peptide moiety determines the enzymespecificity of a substrate. Enzyme activity is measured by the amount ofthe aromatic amine moiety liberated upon hydrolysis of a substrate.Exemplary synthetic substrates are disclosed in U.S. Pat. No. 3,862,011dated Jan. 21, 1975 to Smith, U.S. Pat. No. 4,294,923 dated Oct. 13,1981 to Smith et al.; and U.S. Pat. No. 4,505,852 dated Mar. 19, 1985 toRasnick.

Enzyme specificity is an important criterion for the use of syntheticsubstrates in evaluating proteolytic activities, particularly in theclinical setting. For example, the accuracy and reliability ofdiagnostic tests based on measuring protease activities would beincreased significantly if synthetic substrates were available whichallowed the measurement of a particular protease activity in abiological fluid containing a host of related proteases. That is, suchtests would be significantly improved by the availability of syntheticsubstrates for particular enzymes having little or no cross reactivitywith related enzymes present in the same biological fluid.

The serine proteases are perhaps the best understood class of enzymes intheir catalytic mechanism, and a number of highly specific syntheticpeptide substrates are available for their detection. Cysteineproteases, on the other hand, have not shared the same intensity ofinterest until recently. During the last decade considerable interest incysteine proteases has arisen as evidence of their involvement in anumber of pathological conditions has become more certain. Cathepsins B,H, and L have been linked to inflammation, e.g. Ostensen et al. Clin.Exp. Immunol., Vol. 54, pgs. 397-404 (1983), and protein degradation,e.g. Sutherland et al., Biochem. Biophys. Res. Comm., Vol. 110, pgs.332-338 (1983); McDonald et al., Anal. New York Acad. Sci., Vol. 380,pgs. 178-186 (1982); Quinn et al., Biochem. J., Vol. 172, pgs. 301-309(1978); and Ishura et al., J. Biochem., Vol. 94, pgs. 311-314 (1983).Many tumor cells have been found to secrete a cathepsin B-like cysteineprotease which enables the tumor cells to invade the extracellularmatrix and to metastasize to secondary sites, e.g. Sloane et al., CancerMetastasis Reviews Vol. 3, pgs. 249-263 (1984). Unfortunately suchstudies are limited because currently available synthetic peptidesubstrates used to assay cysteine proteases are often hydrolyzed byserine proteases, making it difficult to assign the observed activitiesto cysteine proteases with confidence.

More specific and selective substrates are necessary to improve orcreate new diagnostic tests based on cysteine protease activities, andto study the specific functions of cysteine proteases in normal andpathological states.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to providesynthetic substrates which are preferentially hydrolyzed by cysteineproteases.

Another object of the invention is to provide synthetic substrates whichare readily hydrolyzed by cysteine proteases, but to which serineproteases show little or no hydrolytic activity.

A further object of the invention is to provide peptide thioneamides asselective substrates for cysteine proteases in biological fluids.

Another object of the invention is to provide a method and kit forassaying the presence of cysteine proteases.

To achieve the foregoing objects, peptide thioneamides are provided asselective synthetic substrates for cysteine proteases. Generally thecompounds of the invention are peptides consisting of one or moreblocked or unblocked amino acids wherein the terminal carboxy of thepeptides are thionated and forms a thioneamide linkage with afluorogenic or chromogenic leaving group. The alpha carbonyls of theremaining amino acids are thionated or unthionated.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention are broadly defined by the formula:

    (AA).sub.n--AAΨ[CS]--W                                 Formula I

wherein:

(AA)_(n) is a blocked or unblocked, thionated or unthionated amino acidwhenever n=1, and (AA)_(n) is a peptide consisting of blocked orunblocked, thionated or unthionated amino acids whenever n is greaterthan one. As used herein the term "thionated or unthionated amino acid"is defined by the following formula: ##STR1## wherein R is defined bythe particular amino acid employed, and Z is oxygen or sulfur. When theterm "thionated" is used herein in reference to an amino acid Z issulfur, and when the term "unthionated" is used Z is oxygen.

n is in the range of 0 to 12, and more preferably in the range of 0 to4.

AAΨ[CS] is a blocked or unblocked amino acid whose alpha carbonyl oxygenhas been replaced with a sulfur atom (that is, the alpha carbonyl is athiocarbonyl), and whose alpha amine forms a peptide bond with the alphacarbonyl of the adjacent amino acid of the (AA)_(n) moiety whenever n isgreater than or equal to one. AAΨ[CS] is defined by the formula:##STR2## wherein R is defined by the particular amino acid employed.

W is a chromogenic or fluorogenic leaving group, such as5-aminoisophthalic acid dimethylester (also referred to herein as AIE),7-amino-4-trifluoromethylcoumarin (also referred to herein as AFC),4-methoxy-2-naphthylamine (also referred to herein as MNA),7-amino-4-methylcoumarin (also referred to herein as AMC),p-nitroaniline (also referred to herein as pNA), cresyl violet,rhodamine, or the like. Preferably W is a fluorogenic leaving group,such as AIE, AFC, MNA, AMC, pNA, or the like. Most preferably W is AIE.

The invention includes salts of the compounds defined by Formula I. Inparticular, such salts include hydrochloride, dihydrochloride,hydrobromide, dihydrobromide, trifluoroacetic, ditrifluoroacetic, andlike salts.

The blocking groups which may be present on AAΨ[CS] or on the amino acidor peptide represented by (AA)_(n) are those well known in the art ofpeptide synthesis. For example, a listing of suitable peptide blockinggroups is found in Gross et al., eds., The Peptides, Vol. 3 (AcademicPress, New York, 1981). The particular choice of blocking group used inthe compounds of the invention depends on several factors, including theblocking group's affect on enzyme specificity, its affect on substratesolubility, and its utility during synthesis. Suitable blocking groupsinclude but are not limited to carbobenzoxy (Cbz), benzoyl,t-butoxycarbonyl (Boc), glutaryl, p-tolylsulfonyl (Tos), methoxysuccinyl(Meosuc), succinyl, glutaryl, and certain D-isomers of naturallyoccurring L-amino acids, including but not limited to D-proline,D-valine, and D-alanine.

Cysteine proteases detectable by compounds of the present inventioninclude, but are not limited to, cathepsins B, H, L, N, S, T, I, J, andK, cancer procoagulant, papain, chymopapain, pyroglutamyl peptidehydrolase, calpain I and II, gamma-endorphin generating endopeptidase,viral cysteine protease, and the like. Selection of the most suitablesubstrate from the set defined by Formula I for detecting a givencysteine protease depends on the protease's specificity requirements.Generally, different cysteine proteases have different activities withrespect to a given peptide bond, and the activities depend critically onthe sequence of amino acids making up the peptide adjacent to thescissle bond. Consequently, picking the best compound of Formula I fordetecting a particular cysteine protease may require some routineexperimentation to determine the optimal peptide moiety for theprotease. Preferably whenever the cysteine protease is cathepsin B thesubstrate is Cbz-Val-Lys-Lys-ArgΨ[CS]-W, Cbz-Arg-ArgΨ[CS]-W,Suc-Tyr-MetΨ[CS]-W, beta-Ala-Tyr-MetΨ[CS]-W, D-Leu-Tyr-MetΨ[CS]-W, orCbz-Ala-Arg-ArgΨ[CS]-W; whenever the cysteine protease is cathepsin Lthe substrate is Cbz-Phe-ArgΨ[CS]-W, Cbz-Arg-ArgΨ[CS]-W,Suc-Tyr-MetΨ[CS]-W, Cbz-Ala-Arg-Arg Ψ[CS]-W, beta-Ala-Tyr-MetΨ[ CS]-W,or D-Leu-Tyr-Met Ψ[CS]-W; whenever the cysteine protease is cathepsin Hthe substrate is ArgΨ[CS]-W, Cbz-Arg-ArgΨ[CS]-W, Suc-Tyr-MetΨ[CS]-W,Cbz-Ala-Arg-ArgΨ[CS]-W, beta-Ala-Tyr-MetΨ[CS]-W, orD-Leu-Tyr-MetΨ[CS]-W; whenever the cysteine protease is pyroglutamylpeptide hydrolase the substrate is PyroGluΨ[CS]-W; whenever the cysteineprotease is cathepsin E the substrate is Cbz-Gly-Gly-ArgΨ[CS]-W;whenever the cysteine protease is cathepsin M the substrate isArgΨ[CS]-W; and whenever the cysteine protease is papain the substrateis Cbz-LysΨ[CS]-W, Meosuc-Phe-Thr(Obzl)Ψ[CS]-W, or Cbz-Phe-ArgΨ[CS]-W.Here the conventional three letter designations for the amino acids havebeen used, e.g. "IUPAC-IUB Commission on Biochemical NomenclatureSymbols for Amino-Acid Derivatives and Peptides Recommendations (1971),"J. Biol. Chem., Vol. 247, pgs. 977-983 (1972).

Generally compounds of the invention are synthesized by first forming anamino acid-leaving group conjugate, which may include suitable blockinggroups. Next, the conjugate is thionated, preferably using Lawesson's,or like reagent, to form a thioneamide, e.g. see Cherkasov et al.,"Organothiophosphorus Reagents in Organic Synthesis," Tetrahedron, Vol.41, pgs. 2567-2624 (1985); Clausen et al., Tetrahedron, Vol. 37, p. 3637(1981); and Yoe et al., Tetrahedron, Vol. 40, pgs. 2047-2052 (1984).Additional compounds of the invention are synthesized by linking athionated or unthionated amino acid to the alpha amine of thethioneamide conjugate, and then to the alpha amine of the thionated orunthionated amino acid just attached, and so on, until a peptidethioneamide of desired length is obtained. The type of thionated aminoacid linked to the leaving group, and the number and ordering of thethionated or unthionated amino acids added thereto in large partdetermine the enzyme specificity for the substrate. As indicated above,any combination of amino acids can be employed to obtain the desiredspecificity. In some cases it may be desirable to employ one or morethionated amino acids in the (AA)_(n) moiety to prevent degradation ofthe substrate by serine proteases which may be present in a samplefluid. Such thionated peptides can be synthesized following theprocedures disclosed in Thorsen et al., Tetrahedron, Vol. 39, pgs.3429-3435 (1983); or in Clausen et al., J. Chem. Soc. Perkin Trans. I,pgs. 785-798 (1984). Accordingly, both of these articles areincorporated by reference.

The method of the present invention for determining the presence of anenzyme in an enzyme-containing analyte comprises contacting the analytewith a substrate which can be hydrolyzed with an enzyme. Such analyte isusually a natural biological fluid, such as blood, serum, urine, tissuehomogenate, or the like, but it can also be a synthetic solution usedfor quality control or as a reference standard. In any case the amountof substrate contacted with the sample fluid must be great enough sothat a detectable fluorescent or colorimetric signal is generated forthe reaction conditions employed. This amount of substrate is referredto herein as an effective amount.

The analyte-substrate mixture is incubated under enzyme hydrolyzingconditions. The pH of the mixture is generally in the range of thenormal physiological environment of the enzyme, and thus can vary fromone enzyme to another. The pH of the mixture is conveniently controlledby mixing the analyte and substrate in an appropriate buffering agent,such as N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES),or the like. In some cases, the concentration of certain divalent metalions, such as Ca⁺⁺, Mg⁺⁺, or the like, must be regulated for properenzyme action. The term "buffering agent" comprehends the inclusion ofagents, e.g. EDTA, to effect such regulation when needed. Thetemperature at which the enzyme hydrolysis is effected is not critical,and may fall within a broad range, provided that the temperature is highenough to ensure enzyme activity, but not too high to cause degradationor other harmful reactions involving the substrate, enzyme, or othercomponents of the mixture.

Fluorimetric or spectrophotometric determination of the liberatedfluorophor or chromophor can be either a rate determination or and endpoint determination. Rate determinations are preferred because they aregenerally more sensitive and precise. In a rate determination, thefluorescence and/or absorption of the substrate-analyte mixture may bedetermined promptly after the analyte is contacted with the substrate.In an end point determination, enzyme hydrolysis is allowed to proceedfor a predetermined length of time, e.g. for about 5 to 60 minutes,preferably for about 15-30 minutes. Such reaction time is selected sothat a sufficient quantity of fluorophor and/or chromophor is releasedas to provide an acceptable degree of accuracy for the assay.

Standard fluorimeters or spectrophotometers are used to make thefluorescence intensity measurements and/or absorption measurements, e.g.see Pearson et al., Clin. Chem., Vol. 27, pgs. 256-262 (1981);Udenfriend, Fluorescence Assay in Biology and Medicine, Vol. I and II(Academic Press, New York, 1961 and 1967); or U.S. Pat. No. 4,388,233,to name just a few references describing appropriate fluorimetric orcolorimetric detection apparatus.

Substrates of the invention are useful for a variety of analyticaltechniques. They can be utilized in biological studies to determine thepresence of cysteine proteases in single cells or tissues, and they areuseful to determine the presence of cysteine proteases in biologicalfluids of clinical importance. The substrates can be used as indicatorsin connection with various chromatographic or electrophoretictechniques. The appropriate substrate can be applied to thechromatographic or electrophoretic medium to indicate the locationand/or density of a previously separated enzyme. Finally, the substratesof the invention can be used to classify enzymes as cysteine proteasesor not.

The following examples serve to illustrate the present invention. Theconcentrations of reagents, choice of temperatures, choice of solvents,and values of other variable parameters are only to exemplify thepractice of the present invention and are not to be considered aslimitations thereof.

EXAMPLE I Synthesis of Cbz-ArgΨ[CS](Mtr)-AIE

Mtr-Cl and Cbz-Arg(Mtr)-OH were synthesized by the method of Fujino etal., Chem. Pharm. Bull., Vol. 29, pgs. 2825-2831 (1981). Here "Mtr"refers to 4-methoxy-2, 3,6-trimethylbenzenesulfonyl, which is employedas a blocking group for the guanidino moiety of arginine. Mixedanhydride coupling method was employed to obtain Cbz-Arg(Mtr)-AIE (R_(f)¹ 1 0.57). Cbz-Arg(Mtr)-OH (5.00 g, 9.62 mmol) was dissolved in 50 ml oftetrahydrofuran (THF) containing 1 eq N-methylmorpholine (NMM), andcooled to -15° C. Isobutyl chloroformate (IBCF) was then added and themixture stirred for 2.5 minutes, after which AIE (2.01 g, 9.62 mmol)dissolved in 20 ml of dimethylformamide (DMF) and cooled to -15° C., wasadded. The mixture was stirred in an ice-methanol bath and slowly warmedto room temperature overnight. The next day, the mixture was evaporatedto an oil which was taken up in EtOAC, and washed twice with 10% citricacid, once with H₂ O, twice with saturated NaHCO₃, and once with brine.The organic layer was dried over MgSO₄ and the solvent evaporated. Theresidue was dissolved in 100 ml CH₂ Cl₂ and precipitated in ether. Theproduct was filtered, dried under reduced pressure over P₂ O₅ and NaOHto give a 47% yield. The material showed a single spot on tlc with R_(f)¹ 0.57.

10 equivalents of Lawesson's reagent(2,4-bis-(4-methoxphenyl)-2,4-dithiooxo-1,3,2,4-dithiadiphosphetanavailable from Fluka) (2.8 g, 7.0 mmol) was added to Cbz-Arg(Mtr)-AIEdissolved in 10 ml of benzene, and the mixture was refluxed for 10 hrs.The reaction was followed by the appearance of a slightly higher R_(f) ¹material on tlc. At the completion of thionation, excess Lawesson'sreagent was filtered off, solvent evaporated, and the product purifiedon a silica gel column. The byproduct of Lawesson's reagent was elutedwith 100% CHCl₃, and then the desired product was eluted with 1%methanol/CHCl₃. After evaporation to a small volume the desired productwas precipitated in ether, filtered, and dried under reduced pressureover P₂ O₅ and NaOH. Tlc showed a single spot with R_(f) ¹ 0.61. (Thecorresponding oxyamide showed R_(f) ¹ 0.57). Yield: 39%.

EXAMPLE II Synthesis of ArgΨ[CS]-AIE.2HBr

Removal of both Cbz and Mtr from Cbz-Arg [CS](Mtr)-AIE was accomplishedsimultaneously by treatment with 31% HBr/Acetic acid for 3 hrs. at roomtemperature. The product was precipitated in ether and purified on aSephadex LH-20 column with 100% methanol as eluent.

EXAMPLE III Synthesis of Cbz-Lys(Boc)Ψ[CS]-AIE

Cbz-Lys(Boc)-AIE was obtained by the mixed anhydride coupling method asdescribed above for Cbz-Arg(Mtr)-AIE. Here "Boc" refers tot-butoxycarbonyl, which is employed as a blocking group for the epsilonamine of lysine. Cbz-Lys(Boc)-AIE (1 g, 1.75 mmol) was refluxed in 10 mlbenzene with 5 equivalents (1.77 g, 4.38 mmol) of Lawesson's reagent for10 hrs. Upon completion of the thionation, the same workup was followedas with Cbz-ArgΨ[CS](Mtr)-AIE, except that the product was eluted with0.5% methanol/CHCl₃ from a silica gel column. The homogeneous materialshowed an R_(f) ¹ 0.89. (The corresponding oxyamide: R_(f) ¹ 0.83)

EXAMPLE IV Synthesis of Cbz-LysΨ[CS]-AIE.HCl

Cbz-Lys(Boc)Ψ[CS]-AIE was dissolved in CH₂ Cl₂ and was treated with anequal volume of saturated HCl/dioxane at 0° C. for 1 hr. The product wasthen precipitated in ether, filtered, and dried under reduced pressureover P₂ O₅ and NaOH. Tlc showed a single spot with R_(f) ² 0.56. (Thecorresponding oxyamide: R_(f) ² 0.48).

EXAMPLE V Synthesis of Cbz-Phe-ArgΨ[CS]-AIE.HBr

The trichlorophenyl ester of benzyloxycarbonylphenylalanine(Cbz-Phe-OTcp) (58.8 mg, 0.123 mmol, 1.5 eq) was combined withArgΨ[CS]AIE.2HBr (50.0 mg, 0.082 mmol) in 3 ml DMF. While stirring,N-methylmorpholine (NMM) (22.6 microliter, 0.205 mmol) and a catalyticamount of 1-hydroxybenzotriazole (HOBT) (1 mg) were added, and thesolution was stirred overnight at room temperature. After evaporation ofthe solvent, the oily material was dissolved in a small amount ofMeOH/CHCl₃ (1:9) and precipitated in ether. The product was thenfiltered and dried under reduced pressure to give a 94% yield. Thematerial showed a single spot on tlc, R_(f) ² 0.71 (the correspondingoxyamide: R_(f) ² 0.64)

EXAMPLE VI Synthesis of Cbz-ArgΨ[CS](Mtr)-AFC and DeprotectedHydrobromide Salt Thereof

Synthesis proceeds as described in Example I, except that AFC issubstituted for AIE to obtain Cbz-Arg(Mtr)-AFC, which is then used inthe thionation step. The hydrobromide salt is obtained as described inExample II.

EXAMPLE VII Synthesis of Cbz-ArgΨ[CS](Mtr)-AMC and DeprotectedHydrobromide Salt Thereof

Synthesis proceeds as described in Example I, except that AMC issubstituted for AIE to obtain Cbz-Arg(Mtr)-AMC, which is then used inthe thionation step. The hydrobromide salt is obtained as described inExample II.

EXAMPLE VIII Synthesis of Cbz-Lys(Boc)Ψ[CS]-AFC and DeprotectedHydrochloride Salt Thereof Synthesis proceeds as described in ExampleIII, except that AFC is substituted for AIE to obtain Cbz-Lys(Boc)-AFC,which is then used in the thionation step. The deprotected hydrochloridesalt is obtained as described in Example IV. EXAMPLE IX Synthesis ofCbz-Lys(Boc)Ψ[CS]-AMC and Deprotected Hydrochloride Salt Thereof

Synthesis proceeds as described in Example III, except that AMC issubstituted for AIE to obtain Cbz-Lys(Boc)-AMC, which is then used inthe thionation step. The deprotected hydrochloride salt is obtained asdescribed in Example IV.

EXAMPLE X Catalysis of Oxyamides and Thioneamides by Trypsin and Papain

Table I below provides a comparison between hydrolysis rates of trypsinand papain acting on two different oxyamides and on their correspondingthioneamides. Trypsin was used at pH 7.5 in a Tris buffer (50 mM), with10 mM CaCl₂, and 12.5% DMSO. Papain was used at pH 6.5 in a thiol buffer(52 mM NaH₂ PO₄, 31 mM dithiothreitol, 2.1 mM EDTA) and 12.5% DMSO.Table I lists the catalytic constants k_(cat) (moles product formed persecond per mole enzyme), K_(M) (M), and k_(cat) /K_(M) (M⁻¹ sec⁻¹) forthe indicated substrates.

                  TABLE I                                                         ______________________________________                                        Substrate         Trypsin    Papain                                           ______________________________________                                        Cbz--Lys--AIE:                                                                k.sub.cat         1.15 × 10.sup.-1                                                                   2.95 × 10.sup.-2                           K.sub.M           1.68 × 10.sup.-4                                                                   2.81 × 10.sup.-4                           k.sub.cat /K.sub.M                                                                              6.86 × 10.sup.2                                                                    1.05 × 10.sup.2                            Cbz--Lysψ[CS]--AIE:                                                       k.sub.cat                    4.00 × 10.sup.-3                           K.sub.M           NO RATE    2.13 × 10.sup.-4                           k.sub.cat /K.sub.M           1.88 × 10.sup.1                            Cbz--Phe--Arg--AIE:                                                           k.sub.cat         9.03 × 10.sup.-1                                                                   3.53 × 10.sup.1                            K.sub.M           5.02 × 10.sup.-4                                                                   8.13 × 10.sup.-4                           k.sub.cat /K.sub.M                                                                              1.80 × 10.sup.3                                                                    4.35 × 10.sup.4                            Cbz--Phe--Argψ[CS]--AIE:                                                  k.sub.cat                    5.35                                             K.sub.M           NO RATE    5.74 × 10.sup.-4                           k.sub.cat /K.sub.M           9.32 × 10.sup.-2                           ______________________________________                                    

Trypsin, a serine protease, showed no measureable rates of productformation for either thioneamide. Papain, a cysteine protease, catalysedproduct formation for both oxyamides and thioneamides.

Table II below shows the selective detection of papain in the presenceof trypsin using Cbz-LysΨ[CS]-AIE. A thiol buffer at pH 6.5 was used (52mM NaH₂ PO₄, 31 mM dithiothreitol, 2.1 mM EDTA) with 2.5% DMSO. Theenzyme concentrations in the assays were 6.85×10⁻⁶ M for papain, and1.28×10⁻⁶ M for trypsin. Substrate concentrations were 2.46×10⁻⁴ M forCbz-Lys-AIE, and 2.53×10⁻⁴ M for Cbz-LysΨ[CS]-AIE. Rates are given interms of moles AIE formed per minute.

                  TABLE II                                                        ______________________________________                                        Enzyme       Cbz--Lys--AIE                                                                              Cbz--Lysψ[CS]--AIE                              ______________________________________                                        Papain       6.03         1.15                                                Trypsin      1.54         0.00                                                Papain and Trypsin                                                                         8.15         1.12                                                ______________________________________                                    

The foregoing disclosure of preferred embodiments of the invention hasbeen presented for purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teaching. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical application, to thereby enable others skilled in the artto best utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto.

We claim:
 1. A compound defined by the formula:

    (AA).sub.n --AAΨ[CS]-W

wherein: (AA)_(n) is a blocked or unblocked, thionated or unthionatedamino acid whenever n=1, and (AA)_(n) is a peptide consisting of nblocked or unblocked, thionated or unthionated amino acids whenever n isgreater than one; n is in the range of 0 to 12 inclusive; AAΨ[CS] is ablocked or unblocked amino acid whose alpha carbonyl oxygen has beenreplaced with a sulfur atom, and whose alpha amine forms a peptide bondwith the alpha carbonyl of the (AA)_(n) moiety whenever n is greaterthan or equal to one; and W is a chromogenic or fluorogenic leavinggroup covalently attached to the carbon atom of the alpha carbonyl ofAAΨ[CS].
 2. The compound of claim 1 wherein (AA)_(n) is a blocked orunblocked, unthionated amino acid whenever n=1, and (AA)_(n) is apeptide consisting of blocked or unblocked, unthionated amino acidswhenever n is greater than one.
 3. The compound of claim 2 wherein W isa fluorogenic leaving group.
 4. The compound of claim 3 wherein n is inthe range of 0 to 4 inclusive.
 5. The compound of claim 4 wherein W isselected from the group consisting of 5-aminoisophthalic aciddimethylester, 7-amino-4-trifluoromethylcoumarin, 4-methoxy-2-naphthylamine, 7-amino-4-methylcoumarin, and p-nitroaniline.6. The compound of claim 5 wherein n=0 and AAΨ[CS] is ArgΨ[CS].
 7. Thecompound of claim 6 wherein W is 5-aminoisophthalic acid dimethylester.8. The compound of claim 5 wherein n=0 and AAΨ[CS] is Cbz-ArgΨ[CS]. 9.The compound of claim 8 wherein W is 5-aminoisophthalic aciddimethylester.
 10. The compound of claim 5 wherein n=0 and AAΨ[CS] isCbz-Lys(Boc)Ψ[CS].
 11. The compound of claim 10 wherein W is5-aminoisophthalic acid dimethylester.
 12. The compound of claim 5wherein n=0 and AAΨ[CS] is Cbz-LysΨ[CS].
 13. The compound of claim 12wherein W is 5-aminoisophthalic acid dimethylester.
 14. The compound ofclaim 5 wherein n=1, (AA)_(n) is Cbz-Phe, and AAΨ[CS] is ArgΨ[CS]. 15.The compound of claim 14 wherein W is 5-aminoisophthalic aciddimethylester.
 16. The compound of claim 5 wherein n=0 and AAΨ[CS] isPyroGluΨ[CS].
 17. The compound of claim 16 wherein W is5-aminoisophthalic acid dimethylester.
 18. The compound of claim 5wherein n=1, (AA)_(n) is Meosuc-Phe, and AAΨ[CS] is Thr(OBzl)Ψ[CS]. 19.The compound of claim 18 wherein W is 5-aminoisophthalic aciddimethylester.
 20. The compound of claim 5 wherein n=3, (AA)_(n) isCbz-Val-Lys-Lys, and AAΨ[CS] is ArgΨ[CS].
 21. The compound of claim 20wherein W is 5-aminoisophthalic acid dimethylester.